Apparatuses for and methods of processing cells and related structures

ABSTRACT

Apparatus for processing life-based organic particles, including particles selected from the list comprising cells, cellular spheroids, tissues, eukaryotes, micro-organisms, organs or embryos, comprises a hollow volume ( 10 ) that (a) is internally divided into at least first ( 14 ), second ( 16 ) and third ( 17 ) sub-volumes by at least two phaseguides ( 12, 13 ) formed inside the volume and (b) includes parts that are relatively upstream and relatively downstream when judged with reference to the movement of a meniscus or a bulk liquid in the volume ( 10 ). The apparatus includes at least first, second and third fluid conduits ( 19, 21, 22 ) connected to permit fluid communication between the upstream exterior of the volume ( 10 ) and a respective said sub-volume ( 14, 16, 17 ); and at least one further conduit ( 24 ) connected to permit fluid communication between the downstream exterior of the volume ( 10 ) and a said sub-volume. The first sub-volume ( 14 ) contains one or more life-based particles supported in or by a gel or gel-like substance; and the second sub-volume ( 16 ) communicates with the first sub-volume so as to permit transport of substances between the first and second sub-volumes ( 14, 16 ) and contains at least one gel or gel-like substance.

The invention relates to apparatuses for and methods of processingcells, cellular spheroids, tissues, eukaryotes, micro-organisms, organs,organic enzymes, gene sequences/chains, proteins or embryos, referred toherein for convenience as “life-based organic particles”. The term“life-based organic particles” potentially includes other structuresthan those listed. The definition of “life-based organic particles” asused herein therefore is not limiting of the scope of the invention asclaimed.

There is strong scientific and commercial interest in the cultivation oflife-based organic particles in laboratory situations closely mimickingthe conditions that such particles would encounter in a complex livingorganism such as but not limited to a mammal.

The reasons for this interest are well known. It has for many years beendesirable to avoid whenever possible the studying of e.g. cells, tissueand organs in vivo. Aside from the fact that this is often veryexpensive and associated with understandably burdensome regulatoryrequirements the results of studies in living creatures may exhibit highvariability from one candidate to the next; and they sometimes presentrisks to the candidates on which the studies are carried out.

Moreover the compounds, reagents and other substances forming thesubject of study may be difficult to administer correctly to locationsin the human or animal body; and the quantities of such substancesrequired for effective dosing using living study samples may be veryhigh.

In recent decades therefore various branches of the scientific communityhave sought to create laboratory environments in which cells, tissues,organs, micro-organs, embryos and in some cases eukaryotes can bestudied away from animal bodies.

Numerous designs of cultivation chamber, tissue scaffold and relatedapparatuses have been created as a result. It has however been foundthat seeking to cultivate e.g. tissues or organs in conditions that donot accurately replicate in-vivo situations can lead to various seriousflaws in experiments and observational activities.

One example, of many, falling in to this category concerns the influenceof flat surfaces of cultivation chambers on cells that in the naturalcondition do not contact such surfaces. Another ever-constant problemconcerns the need to supply living cells with a constantly replenishedsupply of nutrients, oxygen, enzymes and so on; and to remove from thevicinity of cells waste products, metabolytes, respiration products andsimilar substances. In natural environments e.g. a blood supply orlymphatic system may provide these functions but they are very difficultto replicate with accuracy in laboratory equipment intended for cellcultivation.

Many other instances of experimental defect will be known to andrecognised by workers of skill in the art.

Therefore there is a need for apparatuses that accurately mimic in vivoconditions when studies are carried out on life-based organic particlesof the kinds indicated above.

Moreover there is an increasing requirement for tests, assays,reactions, experiments and related activities to be carried out on alarge scale (for example involving many hundreds of samples that arecompleted simultaneously or in accordance with a controlled sequence).Many existing laboratory apparatuses fail to provide these advantages.

It is known to use so-called phaseguides in the construction ofequipment intended to contain and control liquids and liquid-basedsubstances.

A phaseguide may be defined as a structure, in a volume that is to befilled with or emptied of a liquid, that limits the ability of themeniscus of a body of liquid to advance or recede in the volume, therebydefining an interface between the liquid and another substance (i.e.another liquid, or a gas) that is of predetermined shape.

Phaseguides may be constructed in a variety of ways. One techniqueinvolves constructing a sharp edge. Advancement over such a sharp edgerequires a change of the principal radii of a fluid-fluid meniscus,leading to a higher pressure drop over the meniscus thus representing apressure barrier. This concept is also known as “meniscus pinning”.

A typical phaseguide is therefore a three-dimensional structure thatprotrudes into the liquid along the complete length of the meniscus.Pinning of the meniscus on the resulting, elongate protrusion requiressuch additional energy for the liquid meniscus to cross it that theliquid is confined unless additional energy is applied to the body ofliquid.

Another typical phaseguide is a ridge protruding into the bulk material.In this case pinning occurs before the phaseguide.

In addition, the phaseguide may include a usually deliberate location ofweakness at which the energy required to cross the phaseguide is lower.At such a location the liquid may, if the phaseguide is properlydesigned, cross the phaseguide. This deliberate location of weaknessalso defines the “stability” of a phaseguide, which determines the orderor priority of phaseguide overflow when a bulk liquid faces multiplephaseguides simultaneously during meniscus advancement or recession.

A particularly versatile phaseguide is created when the substrate facingthe phaseguide is more hydrophilic than the phaseguide itself. Such animplementation leads to stretching of the meniscus and increases theeffect that angles and their radii have on the stability of aphaseguide.

Thus the phaseguides may, depending on their precise design, eitherconfine a liquid completely; or may permit its advancement or recessiononly at a preferred location so that the liquid follows a chosen path,fills or empties a particular space in the volume, or adopts aparticular shape.

Phaseguides may instead of being constructed as protruding barriers bedefined by areas on an internal surface of a volume that are ofdiffering degrees of wettability. Again such areas may cause arequirement for the input of energy in order to encourage a liquidmeniscus to advance across them.

Numerous designs of phaseguide structure are disclosed in WO2010/085279A2. An understanding of this publication is desirable from thestandpoint of explaining the invention, so the entire content ofWO2010/085279A2 is incorporated herein by reference.

Phaseguides that operate to confine liquids nonetheless may be arrangedto permit controlled crossing, by the liquid, of the barrier representedby the phaseguide; and/or mixing of two liquids confined on oppositesides of a phaseguide or a combination of phaseguides that define aninterposed barrier structure. Arrangements for achieving these effectsare described in WO2010/085279A2, in which the concept of a “confiningphaseguide”, that is of particular utility in embodiments of theinvention described herein, is explained in detail.

According to the invention in a first aspect there is provided apparatusfor processing life-based organic particles including but not limited tocells, cellular spheroids, tissues, eukaryotes, micro-organisms, organsor embryos, the apparatus comprising a hollow volume that (a) isinternally divided into at least first, second and third sub-volumes byat least two phaseguides formed inside the volume and (b) includes partsthat are relatively upstream and relatively downstream when judged withreference to the movement of a meniscus or a bulk liquid in the volume,the apparatus including at least first, second and third fluid conduitsconnected to permit fluid communication between the upstream exterior ofthe volume and a respective said sub-volume; and at least one furtherconduit connected to permit fluid communication between the downstreamexterior of the volume and a said sub-volume, the first said sub-volumecontaining one or more life-based particles supported in or by a gel orgel-like substance; and the second sub-volume communicating with thefirst sub-volume so as to permit transport of substances between thefirst and second sub-volumes and containing at least one gel or gel-likesubstance.

An advantage of this arrangement is that it becomes possible to createtruly realistic cultivation environments in laboratory situations whilealso providing the possibility of performing very large-scale assays andtrials as described above. The apparatus of the invention thereforeaddresses the drawbacks and requirements set out herein.

Optional aspects of the invention are defined in the dependent claims.

The invention is also considered to reside in a method of processingcells, cellular spheroids, tissues, eukaryotes, micro-organisms, organsor embryos comprising the steps of:

-   -   a. charging the first volume of apparatus in accordance with the        invention as claimed herein with a quantity of a gel or gel-like        substance containing or supporting one or more cells, cellular        spheroids, tissues, eukaryotes, micro-organisms, organs or        embryos;    -   b. permitting or causing gelation of the gel or gel-like        substance;    -   c. charging at least the second sub-volume with a gel or        gel-like substance; and    -   d. causing or permitting fluid communication between the first        and second sub-volumes.

A further method according to the invention includes the steps of:

-   -   a. charging the first volume of apparatus according to claim 15        hereof with a quantity of a gel or gel-like substance containing        or supporting one or more cells, cellular spheroids, tissues,        eukaryotes, micro-organisms, organs or embryos;    -   b. charging at least the second sub-volume with a gel or        gel-like substance; and    -   c. causing or permitting fluid communication between the first        and second sub-volumes.

There now follows a description of preferred embodiments of theinvention, by way of non-limiting example, with reference being made tothe accompanying drawings in which:

FIG. 1 is a schematic, horizontally sectioned view of one form ofapparatus in accordance with the invention;

FIG. 2 is a view similar to the FIG. 1 illustration of anotherembodiment of apparatus according to the invention;

FIGS. 3 a and 3 b shows two states of filling of a further embodiment ofapparatus according to the invention;

FIGS. 4 a-4 c show in schematic form three states of filling of afurther, more complex apparatus according to the invention; and

FIGS. 5 a-5 c are similar views to FIGS. 4 a-4 c showing theconstruction and use of an apparatus according to the invention that issuitable for so-called “one shot” perfusion of life-based organicparticles in a complex array of cultivation chambers.

Referring to the drawings, in FIG. 1 a hollow cuboidal volume 10constructed in any of a number of ways known in the art of phaseguidechamber technology for example as described in WO2010/085279 A2 is inthe embodiment shown internally divided by first, second and thirdphaseguides 11, 12, 13 into four stripe-like sub-volumes 14, 16, 17, 18.

In the illustrated embodiment of the invention the sub-volumes 14, 16,17, 18 are shown extending from one end of the interior of the volume 10to the other in parallel with one another. The sub-volumes arerectangular in plan view and would have a vertical extent in a directionperpendicular to those visible in FIG. 1.

The volume 10 and the sub-volumes 14, 16, 17, 18 may adopt other shapesand forms as required. The phaseguides 11, 12, 13 need not follow thestraight line patterns shown and may be curved or part-polygonal forexample. An enormous variety of phaseguide, volume and sub-volumeshapes, orientations, sizes and arrangements is possible within thescope of the invention, with the FIG. 1 arrangement being employed asmerely an illustrative example.

It is preferable that the interiors of the sub-volumes may be observedin some experiment-based fashion, and especially optically. To this endthe sub-volumes may include transparent or translucent walls or windowsas is known to the worker of skill. In some embodiments one or more ofthe sub-volumes may be open on e.g. an in-use upper side, but in themajority of practical embodiments the volume 10 would be substantiallyclosed in order to isolate the experimental environment from ambientconditions that might otherwise induce evaporation, temperature changesor contamination.

The two sub-volumes 14, 16 extending respectively to either side of theend-to-end centre line of the volume 10 occupied by phaseguide 12 areherein designated the first and second sub-volumes. The sub-volumes 17,18 lying horizontally outwardly of the first and second sub-volumes 14,16 are designated third and fourth sub-volumes respectively.

Each of the sub-volumes 14, 16, 17, 18 includes connected thereto at oneend 10 a designated the upstream end when considering filling of thesub-volumes with fluids a respective fluid conduit 19, 21, 22, 23. Thedesigns of the conduits may vary significantly within the scope of theinvention but their main function is to permit fluid communicationbetween sources of fluids (not shown) located externally of thesub-volumes; and the insides of the sub-volumes 14, 16, 17, 18.

At the opposite, herein downstream, (when judged with reference tofilling of the sub-volumes 14, 16, 17, 18) end 10 b of the volume 10each of the third and fourth sub-volumes 16, 17 is connected to afurther respective conduit 24, 26.

The conduits 19, 23 function to supply nutrients, oxygen, medium, plasmaand/or a range of further fluids to life-based organic particles in thesub-volumes 14, 16 as described below; and the further conduits 24, 26are intended to transport depleted medium, depleted buffer, depletedplasma, waste products, respiration products, metabolytes and so on awayfrom such particles.

To this end the first sub-volume 14 contains a gel or gel-like substance29 that supports one or more life-based particles 27 illustratedschematically in FIG. 1. The gel 29 may be chosen to provide a physicalsupport for the particles 27 and also to provide a correct biochemicalenvironment for cultivation and/or viability of the particles for theduration of a study.

Examples of gels or gel-like substances that are suitable for use in thesub-volumes of the invention include but are not limited to basalmembrane extract, human or animal tissue, cell culture-derivedextracellular matrices, animal tissue-derived extracellular matrices,synthetic extracellular matrices, hydrogels, collagen, soft agar, eggwhite and commercially available products such as Matrigel®.

The second sub-volume 16 contains a second gel or gel-like substance, ora non-gel liquid, 31 that may be similar to substance 29 or may differtherefrom, depending on the requirements of the experiment. Substance 31may contain chemicals, nutrients or pharmaceuticals the effect on theparticles 27 it is required to investigate, or (for example) furtherlife-based particles 28 represented schematically in FIG. 1. A widevariety of material choices is possible within the scope of theinvention.

The phaseguide 12 separating sub-volumes 14 and 16 from one anothertypically in accordance with the invention is a confining phaseguidesuch as those described in WO2010/085279A2. A characteristic of suchphaseguides is that they can be designed to permit controlled mixing,diffusion or perfusion of the substances in the adjacent sub-volumes 14,16.

Thus as just one example of use of the apparatus of FIG. 1 the gel, etc,31 in second sub-volume 16 may contain cells 28 that interact with e.g.cells 27 in first sub-volume 14. The combination of cells 27 in firstsub-volume 14 and cells 28 in sub-volume 16 thereby are combined in amanner that accurately replicates an in vivo situation.

As another example of use of the apparatus of FIG. 1 the gel, etc, 31 insecond sub-volume 16 may contain a chemical compound that affects e.g.cells 27 in first sub-volume 14 under investigation. The cells 27 infirst sub-volume 14 thereby may become dosed in a manner that accuratelyreplicates an in vivo situation, with the cells supported in a gel orgel-like substance the density, viscosity composition of which mimic themedia in which the cells would naturally exist.

Another option is for the transport of life-based particles from thefirst sub-volume into the second sub-volume to occur, as a result of theexistence of a gradient including but not limited to a gradient inchemical concentration, chemical composition, temperature, pressure,electric field, light, nutrients, oxygen, gel density and gelcomposition

The arrangement of the invention therefore evidently solves some of thelong-standing drawbacks of the prior art.

Maintaining the viability of cells in an experimental situation is oftendifficult. The purpose of the sub-volumes 17 and 18 is to provide forthe transport, in a continuous flow via conduits 19 and 24 (in the caseof sub-volume 18) and 23 and 26 (in the case of sub-volume 17), ofnutrients, oxygen, carbon dioxide, growth factors, other proteins,signalling molecules, compounds, further cells and the like into thevicinity of the sub-volumes 14, 16; and the transport away of wasteproducts, metabolytes and the like. This activity is indicated by thearrows in FIG. 1. The arrangement of the invention provides forconsiderable improvements in cell, etc, viability than prior artarrangements.

To this end the conduits 19, 24, 23, 26 could be connected for examplein liquid pumping/supply circuits the nature and operation of which willbe known to the worker of skill.

The sub-volumes 17, 18 need not contain the same substances; and in manycases the substances transported in the two sub-volumes 17, 18 willdiffer from one another.

The phaseguides 11, 13 in view of the foregoing are respectivelyarranged to permit or promote the transport of liquids or othersubstances into and/or out of the sub-volumes 14, 16 from/to thesub-volumes 18, 17. These phaseguides therefore also could be confiningphaseguides of the kind described in WO2010/085279A2.

Another mode of use of the apparatus of the invention might relate tothe cultivation, etc, of cells in proximity to one another (without arequirement for transport of life-based particles from one sub-volume toanother). It is apparent that the FIG. 1 apparatus readily could beemployed in this manner, with the cells, etc, 27, 28 maintained in therespective first 14 and second 16 sub-volumes for the duration of anexperiment.

Examples of cell types that may be co-cultured in the ways describedherein, using the apparatus of the invention, include co-cultures ofe.g. cancer cells with cancer associated fibroblasts (CAFs); liver cellswith any other cells or tissues; cartilage with osteoblast; various celltypes present in colon and intestinal cells; endothelial blood vesselcells in combination with any other cell type, including such cellcombinations derived from the brain functioning as ex-vivo blood-brainbarriers; skin cells; cells involved in lung tissue; and combinations ofcells excreting hormone or other signaling factors and target tissues.

The co-cultures may contain gradients of differentiating cells, forexample differentiation gut crypt cells; and embryonic cells such asendo-, meso- and ectoderm, stem cells. For the avoidance of doubthowever to the extent that this patent application gives rise to rightsin a jurisdiction that does not support the grant of patent rights inrespect of human embryonic stem cells such cells obtained from humanembryos; their use; and treatments of human embryos are excluded fromthe scope of protection under the claims hereof.

Concentration gradients of hormones, nutrients growth factors, gases orany cell-cell, tissue-tissue or organ-organ signaling factors canthrough use of the invention be established across cultured cells, thusexposing the cells to said signaling factors, hormones, nutrients orgases according to their position in the established gradient.

In another arrangement according to the invention the sub-volumes 17, 18could contain chemicals the flow rates and concentrations of which aresuch as to establish a concentration gradient from one side of the firstsub-volume 14 to the other (as viewed in FIG. 1) or, through judiciousarrangement of the fluid flow rates in sub-volumes 17, 18, from one endof the first sub-volume 14 to the other.

In particular in this regard at low flow rates in sub-volumes 17, 18 itbecomes possible to maintain concentration gradients across thesub-volume 14 and if present (see below) the sub-volume 17 that aregenerally constant from one end of the sub-volumes 14, 16 to the other.At higher rates of flow the concentration gradient across thesub-volumes 14, 16 may be arranged to vary from one end of thesub-volumes to the other. As an example of this, a high concentrationgradient may be arranged to exist adjacent the conduits 21, 22 and alesser concentration gradient at the opposite end of the sub-volumes 14,16.

Another possibility is to arrange for the flow of liquid in thesub-volumes 17, 18 respectively to be in opposite directions, such thatone of the arrows in FIG. 1 would be reversed. As a result particulargradient profiles may be developed in the sub-volumes.

Another possibility is to arrange for the flow of liquid in thesub-volumes 17, 18 not to be alongside the first and second sub-volumeregions, and instead passing through the sub-volume regions.

Yet another option is to arrange for the liquid in sub-volume 17 andsub-volume 18 to be static during an experiment. As a result aconcentration gradient may be established in the sub-volumes 14, 16 thatalters over time until an equilibrium exists.

If in the alternative only one of the sub-volumes 17, 18 contains astatic liquid the concentration of a compound in the other sub-volume17, 18 would alter over time as its contents become increasingly diluteowing to material transport across the phaseguides illustrated.

The sub-volumes 17, 18 could in some embodiments within the scope of theinvention contain gases or sets of immiscible liquids.

A greater number of the sub-volumes could be defined within the volume10 if desired. Moreover the insides of the various sub-volumes couldthemselves be sub-divided for example by one or more phaseguides of the“contour” type that as described in WO2010/085279A2 can assure aparticular pattern of filling of a volume with a fluid. One particularoption that is thereby made possible is the stratification of substancesinside one or more of the sub-volumes.

The sub-volume 17 and associated conduits 23, 26 may be omitted fromsome embodiments of the invention.

The foregoing arrangement therefore represents the most rudimentaryversion of the invention that is presently known to the inventors.

Use of the apparatus of FIG. 1 typically would involve firstly supplyinga gel or gel-like substance, containing life-based particles ofexperimental interest, via conduit 21 into sub-volume 14. Thereafter itwould be necessary to await gelation of the gel before carrying out asimilar activity in respect of sub-volume 16, since otherwise the bodyof gel in sub-volume 14 may not be sufficiently stable as to permit thecommencement of experimentation.

Once stable conditions are established in the sub-volumes 14, 16 theflow of fluids by way of conduits 19, 24, 23, 26 may commence inaccordance with the protocol of the experiment of interest.

Referring now to FIG. 2 there is shown an apparatus that is similar tothe FIG. 1 apparatus but is configured to promote the transport oflife-based particles, and especially cells 27, from the first sub-volume14 into the second sub-volume 16. Such transport is signified by arrowsin FIG. 2.

In the embodiment of FIG. 2 the second sub-volume 16 is shown devoid oflife-based particles but this need not necessarily be the case.

A variant on this arrangement is one in which an additional sub-volumemay be defined inside volume 10 and allowing for the cells 27 insub-volume 14 to grow or migrate into one or both of two adjacentsub-volumes, or from outer sub-volumes towards each other in innersub-volumes. This migration may be a result of a gradient including butnot limited to a gradient in chemical concentration, chemicalcomposition, temperature, pressure, electric field, light, nutrients,oxygen, gel density and gel composition. This may result in productionof e.g. a scratch assay, invasion assay, migration assay or woundhealing assay.

Use of the FIG. 2 apparatus is similar to that of FIG. 1, in that itinvolves the sequential injection of gel-like substances into thesub-volumes 14, 16 with a need for a gelation delay between respectivegel injections.

The requirement in use of the apparatus as described above for one gelor gel-like substance to gelate before it becomes possible to add afurther such substance to the apparatus may be inconvenient in somesituations. A solution to this difficulty is provided by the arrangementof FIGS. 3 a and 3 b in which an additional phaseguide 32 extendsparallel to phaseguide 12 from one end of the inside of volume 10 to theother.

Such a phaseguide may be arranged to confine the gel or gel-likesubstance 29 in sub-volume 14, without disturbing the profile in which agel or gel like substance is distributed in the volume, while the gel orgel-like substance 31 is being filled into sub-volume 16. This avoidsthe need for any delay between the filling steps described above.

FIG. 3 a illustrates the state of the apparatus during filling of thesecond sub-volume 16 in this way. In FIG. 3 a therefore the gel orgel-like substance 31 does not yet occupy the entirety of thesub-volume; and an exemplary cell 28′ is visible in the conduit 22 onits way into the sub-volume 16.

In FIG. 3 b the sub-volume 16 is filled and all the cells 28 are insideit.

The phaseguide 32 optionally includes a so-called “engineered overflow”point 33 that for convenience is shown at one end of the phaseguide 32but could in reality be at any chosen location along its length (andincluding at one extreme end as shown). Such a feature 33 may be used toinduce or promote controlled mixing of the substances in the sub-volumes14, 16 (or as described herein transport of particles from onesub-volume to another).

Any of the phaseguides disclosed herein could optionally include one ormore such engineered overflow features 33.

FIG. 4 shows a third basic version of the invention in which an array 36of volumes 10, each of which in the embodiment shown includes three ofthe sub-volumes 14, 16, 17, 18 (although more sub-volumes may be presentas desired) is interconnected by fluid-conveying conduits 37.

The central sub-volume 14 of each volume 10 of the array contains e.g. agel supporting life-based particles. Adjacent to that gel is a perfusionflow in sub-volume 16. The perfusion flow addresses multiple volumes inparallel, starting from the same origin signified schematically by arrow37 a. It is possible then to perfuse multiple examples of thesub-volumes 14 using a “one-shot perfusion” technique in which thesingle origin simultaneously perfuses a large number of sub-volumes.

The conduits 37 of the perfusion network are arranged such that apartition of perfusion flow is never flushed along two volumes insequence. This is important in order to avoid contaminating one volume10 or sub-volume 14 with aspects of another such volume or sub-volume.

A second preferred aspect of the perfusion network is that all flowpaths have the same length. This is important in order to guarantee anequal perfusion speed in all chambers.

The challenge of filling such a balanced flow network is solved with thehelp of phaseguides 38, that typically are as described inWO2010/085279A2. Each volume has downstream of it in a section of theconduit 37 a phaseguide 38 that assures filling of all volumes. In orderto completely fill the downstream conduit 37 c that is downstream of thevolume 10 in question.

The phaseguide most distal to the downstream end of the commondownstream conduit 37 c is of lower stability than the other phaseguidesalong the common downstream conduit 37 c. Overflow of this weaker (i.e.less stable) phaseguide therefore occurs first and filling of thedownstream conduit commences at its distal part. This sequence offilling is illustrated by FIGS. 4 a-4 c, with the dark shadingindicating the progressive filling of different parts of the conduitnetwork 37 with perfusate. For the avoidance of doubt, relativelyupstream and downstream directions herein are defined with reference tothe direction of advancement of fluid in the conduits 37, as signifiedby arrow 37 a.

Optionally, the common downstream conduit 37 c can be omitted and eachvolume have its own perfusion outflow unit.

Also optionally, in the case of a weak gel, the pressure on the gelduring initial filling can be relieved by introducing a contourphaseguide, similar to phaseguide 37 in FIG. 3.

FIGS. 5 a-5 c show a more complex one-shot perfusion flow network, inwhich the conduit geometry of FIG. 4 is embedded in a network ofconduits 39, 41 that are higher in hierarchy. In this case additionalphaseguides are needed to also fill the common downstream conduit of thehigher hierarchy. This is done similar with a similar phaseguideconfiguration as in FIG. 4, with the exception that the phaseguide oflower stability needs to be of higher stability than the phaseguide oflower stability of the common downstream conduit that is lower inhierarchy.

In other words the hierarchy of the respective conduit/volumearrangement determines the stability of the phaseguides that are neededin it in order to assure a correct filling order even in the case of acomplex hierarchy as represented by FIG. 5.

In practical situations the arrays of FIGS. 4 and 5 may be significantlymore extensive than the illustrations, which are simplifications forease of presentation. Careful choice of phaseguide stabilities permitssuccessful operation of even highly extensive networks containing manytens or hundreds of the volumes 10.

Any of the volumes 10 described herein may be associated with one ormore excess flow volumes. These are volumes that are connected to theconduits described herein by respective branch conduits.

The purpose of an excess flow volume is to ensure filling of a volume 10by the correct quantity of fluid, even when a greater quantitypotentially could enter it by reason of being present in the associatedconduit responsible for filling of the volume.

Any excess flow volume of this kind would include at or near itsentrance a phaseguide the stability of which is chosen in order toassure that filling of the volume 10 completes before any excess flowpasses into the excess flow volume.

The excess flow volumes are described in more detail in patentapplication no GB 1103917.9, and typically would include one ore morevents for venting gas in the excess flow volume that is expelled duringfilling with excess liquid as described.

It is not necessary, in a complex array such as that outlined above, foreach volume 10 to be associated with its own excess flow volume. On thecontrary, each conduit of the network representing a hierarchical levelin the array for instance may have an excess flow volume connected to itby a branch conduit (this being in many cases a sufficient amount ofexcess flow capacity in the network). As indicated the choice of thestabilities of the phaseguides typically at the entrances to the excessflow chambers determines that they fill in a preferred order, followingfilling of the volumes proper 10, in the event of an excess flow beingpresent.

Overall the apparatus and methods of the invention represent highlysignificant advances in the practical applications of so-calledmicrofluidics techniques to topics such as cell cultivation andexperimentation.

The listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat the document is part of the state of the art or is common generalknowledge.

1. Apparatus for processing life-based organic particles includingparticles selected from the list comprising cells, cellular spheroids,tissues, eukaryotes, micro-organisms, organs or embryos, the apparatuscomprising a hollow volume that (a) is internally divided into at leastfirst, second and third sub-volumes by at least two phaseguides formedinside the volume and (b) includes parts that are relatively upstreamand relatively downstream when judged with reference to the movement ofa meniscus or a bulk liquid in the volume, the apparatus including atleast first, second and third fluid conduits connected to permit fluidcommunication between the upstream exterior of the volume and arespective said sub-volume; and at least one further conduit connectedto permit fluid communication between the downstream exterior of thevolume and a said sub-volume, the first said sub-volume containing oneor more life-based particles supported in or by a gel or gel-likesubstance; and the second sub-volume communicating with the firstsub-volume so as to permit transport of substances between the first andsecond sub-volumes and containing at least one gel or gel-likesubstance.
 2. Apparatus according to claim 1 wherein the thirdsub-volume communicates with at least the first sub-volume so as topermit transport of substances between the first and third sub-volumes,and wherein the third sub-volume contains a perfusate.
 3. Apparatusaccording to claim 2 wherein the perfusate is flowing inside the thirdsub-volume.
 4. Apparatus according to claim 1, wherein the secondsub-volume contains one or more cells, cellular spheroids, tissues,eukaryotes, micro-organisms, organs or embryos supported in or by thegel or gel-like substance.
 5. Apparatus according to claim 4 wherein theone or more cells, cellular spheroids, tissues, eukaryotes,micro-organisms, organs or embryos in the second sub-volume differ fromthe one or more cells, cellular spheroids, tissues, eukaryotes,microorganisms, organs or embryos in the first sub-volume.
 6. Apparatusaccording to claim 1, including a third phaseguide defining a fourthsub-volume inside the volume.
 7. Apparatus according to claim 6 whereinthe fourth sub-volume communicates with at least the first sub-volume soas to permit transport of substances between the first and fourthsub-volumes, and wherein the fourth sub-volume contains a perfusate. 8.Apparatus according to claim 7 wherein the perfusate is flowing insidethe third sub-volume.
 9. Apparatus according to claim 6 wherein thefourth sub-volume contains a fluid substance the effect of which on oneor more cells, cellular spheroids, tissues, eukaryotes, micro-organisms,organs or embryos in the first volume is required to be investigated.10. Apparatus according to claim 6 wherein the third and fourthsub-volumes contain fluid substances that give rise to a concentrationgradient in at least the first sub-volume.
 11. Apparatus according toclaim 9 wherein the fluid substance is or includes a liquid orliquid-based substance.
 12. Apparatus according to claim 6 wherein thefourth sub-volume contains a gas.
 13. Apparatus according to claim 1,wherein at least the first sub-volume includes one or more phaseguidesthat stratify the gel or gel-like substance therein.
 14. Apparatusaccording to claim 1, including connected to at least the first fluidconduit connected to the first sub-volume a further volume; and theapparatus including one or more phaseguides that control thetransmission of liquids or liquid-based substances to the further volumesuch that on charging of the first volume with a predetermined quantityof a gel or gel-like substance any gel or gel-like substance in excessof the predetermined quantity enters the further volume.
 15. Apparatusaccording to claim 1, including at least one contour phaseguide in thevolume between the first and second sub-volumes and arranged to maintaina predetermined shape of a boundary of a gel or gel-like substance in atleast the first sub-volume.
 16. A method of processing cells, cellularspheroids, tissues, eukaryotes, microorganisms, organs or embryoscomprising the steps of: a. charging the first volume of apparatusaccording to claim 1, with a quantity of a gel or gel-like substancecontaining or supporting one or more cells, cellular spheroids, tissues,eukaryotes, micro-organisms, organs or embryos; b. permitting or causinggelation of the gel or gel-like substance; c. charging at least thesecond sub-volume with a gel or gel-like substance; and d. causing orpermitting fluid communication between the first and second sub-volumes.17. A method of processing cells, cellular spheroids, tissues,eukaryotes, microorganisms, organs or embryos comprising the steps of:a. charging the first volume of apparatus according to claim 15 with aquantity of a gel or gel-like substance containing or supporting one ormore cells, cellular spheroids, tissues, eukaryotes, micro-organisms,organs or embryos; b. charging at least the second sub-volume with a gelor gel-like substance; and c. causing or permitting fluid communicationbetween the first and second sub-volumes.